The IGF2BP3/Notch/Jag1 pathway: A key regulator of hepatic stellate cell ferroptosis in liver fibrosis

ABSTRACT Introduction Liver fibrosis is primarily driven by the activation of hepatic stellate cells (HSCs), which involves various epigenetic modifications. Objectives N 6‐methyladenosine (m6A), the most prevalent RNA modification in eukaryotic cells, influences numerous physiological and pathological processes. Nevertheless, the role of insulin‐like growth factor 2 mRNA‐binding protein 3 (IGF2BP3), a reader gene mediating m6A modifications, in liver fibrosis remains unclear. Methods and results This study demonstrated that IGF2BP3 knockout reduces liver fibrosis by promoting HSC ferroptosis (FPT) and inactivating HSCs. Multi‐omics analysis revealed that HSC‐specific IGF2BP3 knockout decreased m6A content in Jagged1 (Jag1), a key component of the Notch signalling pathway. Furthermore, IGF2BP3 deficiency significantly reduced the expression of hairy and enhancer of split‐1 (Hes1), a transcription factor in the Notch/Jag1 signalling pathway, with mRNA levels declining to 35%–62% and protein levels to 28%–35%. Additionally, it suppressed glutathione peroxidase 4 (GPX4) (decreased to approximately 31%–38%), a negative regulator of FPT, thereby facilitating HSC FPT progression and reducing profibrotic gene expression. Conclusion These findings uncover a novel IGF2BP3/Notch/Jag1 signalling pathway involving HSC FPT, suggesting promising targets for ameliorating liver fibrosis. Key Points/Highlights IGF2BP3 deficiency inactivates Jag1 signalling. IGF2BP3 deficiency‐mediated m6A modifications promote HSC ferroptosis. IGF2BP3 inhibition facilitates ferroptosis in HSCs via the Hes1/GPX4 axis. IGF2BP3 deficiency inactivates Jag1/Notch1/3/Hes1 signalling pathway inactivation, leading to the decrease in GPX4, which contributes to HSC ferroptosis.


INTRODUCTION
Liver fibrosis, a pathological response to chronic liver injury, is marked by the activation of hepatic stellate cells (HSCs) and the deposition of extracellular matrix components, ultimately disrupting liver architecture. 1Without intervention, liver fibrosis inevitably progresses to cirrhosis and hepatocellular carcinoma (HCC).Activated HSCs are considered the principal drivers of liver fibrosis due to their excessive production of the extracellular matrix. 2Therefore, suppressing HSC activation is a critical objective in combating liver fibrosis.
The N 6 -methyladenosine (m 6 A) modification of RNA plays a vital regulatory role in the gene expression through three types of effector proteins: writers, readers and erasers. 3The methyl group is added to the adenosine base by a multi-subunit complex ("writer"). 4This modification can be "read" by RNA-binding proteins, primarily those from the YT521-Bhomologydomain (YTH) family and the insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs) family, and can be reversed by "eraser" proteins. 5Dysregulation of the "reader" IGF2BP3 affects m 6 A levels and disrupts various biological behaviors.For instance, in nasopharyngeal carcinoma, IGF2BP3 promotes proliferation and metastasis by altering m 6 Amodified KPNA2 stability. 6IGF2BP3 also plays a role in the pathological and physiological regulation of HCC. 7 However, its involvement in liver fibrosis remains unclear.
Ferroptosis (FPT) is a novel form of iron-dependent cell death characterised by lipid peroxidation. 8Cells undergoing FPT exhibit distinctive shapes, biochemistry and genetics.Morphologically, FPT is identified by decreased mitochondrial volume, increased bilayer membrane density and the disappearance of mitochondrial cristae. 9ecent research has demonstrated a link between m 6 A modification and FPT in HCC. 10 Consequently, we hypothesise that targeting m 6 A modification to induce FPT in HSCs could be an effective strategy for FPT-based treatments.
This study explored the role of IGF2BP3 in liver fibrosis and uncovered a novel signalling pathway and molecular mechanisms of FPT in liver fibrosis.Our findings indicate that the m 6 A reader IGF2BP3 recognises Jag1 m 6 A binding sites to inhibit the Notch pathway and inactivate downstream molecule Hes1 to regulate GPX4, leading to HSC FPT.HSC-specific deletion of IGF2BP3 enhanced HSC FPT and impeded their activation, thereby reducing liver fibrosis through the IGF2BP3/Notch/Jag1 signalling pathway.Thus, we identified IGF2BP3 as a novel regulator of liver fibrosis with significant translational potential for treating this condition.
Liver fibrosis was induced using carbon tetrachloride (CCl 4 ), administered biweekly for 6 weeks.The control group (n = 6) received equivalent volumes of olive oil injections.To induce the non-alcoholic steatohepatitis (NASH) model, mice (n = 6) were fed a high-fat diet (HFD) for 16 weeks, whilst the control group (n = 6) was fed a normal diet.Post-experiment, blood, liver tissue and primary HSC samples were collected for analysis.

Human cirrhosis biopsy specimens
This study involved collecting biopsy samples and sera from 30 patients with cirrhosis and 30 healthy individuals at the First Affiliated Hospital (FAH) of Wenzhou Medical University.

Serum alanine aminotransferase and aspartate aminotransferase assay
Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice were measured using assay kits.

Quantitative real-time PCR
Total RNA was extracted from primary HSCs, LX2 cells, and liver tissues using TRIzol reagents.The relative mRNA levels were quantified via the 2 −ΔΔCT method, with β-actin as the internal control.Primer sequences for quantitative real-time PCR (qRT-PCR) are listed in Table S1.

Pathological evaluation of liver
Liver tissues were stained with hematoxylin and eosin (HE), Sirius Red, and Masson stains. 11

Immunohistochemistry
Following previously described protocols, 12 samples were fixed in formalin, blocked with 10% bovine serum albumin, incubated with antibodies.The stained regions were examined under a Carl Zeiss microscope.Antibody details are provided in Table S2.

Isolation and culture of primary hepatic stellate cells
Following previously described protocols, 13 primary HSCs were isolated from control and IGF2BP3 cKO mice and the cells purity exceeding 98%.

Cell viability assay
Cell viability was assessed using the Cell Counting Kit-8 (CCK8) assay.In brief, 5 × 10 3 primary HSCs and 8 × 10 3 LX2 cells were seeded in 96-well plates and subjected to the indicated treatments.Optical density at 450 nm was measured after 3 h using a microplate reader.

Western blot
Proteins were separated using 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and subsequently transferred to a polyvinylidene difluoride membrane and then incubated with antibodies.Details of the antibodies used are listed in Table S2.

Construction of cell lines with RNA knockdown and overexpression
Lentiviruses were constructed and packaged by GenePharma (Shanghai, China).Primary HSCs and LX2 cells were seeded in 24-well plates for 24 h, achieving a cell fusion rate of 40%−60%.The lentivirus stock solution was diluted with 10% FBS to a concentration yielding the highest infection efficiency, determined to be 1:10.The puromycin concentration was maintained for over 120 h to select the optimal concentration for complete cell elimination, identified as 5 µg/mL.The resulting cells were collected for further analysis.

RNA-sequencing profiling
Total RNA was isolated from primary HSCs of control and IGF2BP3 −/− mice.Following quality assessment, RNA sequencing libraries were constructed and sequencing was performed using the Illumina NovaSeq 6000 platform.Differential expression analysis employed a threshold of |Log 2 fold change| > .585and adjusted p < .05.

m 6 A RNA immunoprecipitation-sequencing
Primary HSCs were isolated and cultured in vitro for spontaneous activation.m 6 A RNA immunoprecipitation (RIP) was performed as previously described. 14An m 6 A antibody was used to enrich RNA fragments.Intact mRNA was isolated and fragmented purified mRNA into approximately 300-nt fragments.Sequencing libraries were constructed and sequencing on the Illumina HiSeq 6000 platform.

mRNA m 6 A level
Purified mRNA was immobilised in assay wells, and total RNA was bound to strip wells.With the enhanced signal measured via absorbance using a microplate spectrophotometer, followed by colorimetric quantification.

Immunofluorescence
Liver tissues were fixed, embedded and sectioned into 3µm-thick slices.Primary HSCs and LX2 cells were fixed, permeabilised, blocked, and incubated with antibodies.Details of the antibodies used are provided in Table S2.

Transmission electron microscopy
Cells were fixed, embedded, dehydrated and treated with uranyl acetate and lead citrate.Representative images were captured using transmission electron microscope (TEM).

Iron measurements
As previously described, 16 an acidic buffer was added to the iron assay buffer to liberate the iron.After removing insoluble material, a microplate fluorometer determined the iron concentration.

Lipid reactive oxygen species assay
As previously described, 16 cells were treated with Ferrostatin-1 (Fer-1) or RAS-selective lethal 3 (RSL3) for 24 h, followed by incubation with BODIPY-C11 dye and the amount of reactive oxygen species (ROS) was measured.

Detection of malondialdehyde and glutathione
Malondialdehyde (MDA) and glutathione (GSH) levels were quantified using activity assay kits.The contents of MDA and GSH were measured at 450 and 405 nm, respectively, using a microplate fluorometer.

Chromatin immunoprecipitation
GENMED's chromatin immunoprecipitation (ChIP) Enzymatic Chromatin IP kit was employed for ChIP.Cells were lysed with SDS buffer, and DNA was fragmented into 100−500 bp pieces via ultrasound.DNA fragments were then immunoprecipitated using specific antibodies or normal mouse IgG, and the enriched sequences were analysed by qRT-PCR.

Luciferase assay
The luciferase activity was measured as previously described. 17GPX4-WT and GPX4-MUT plasmids were co-transfected with Hes1 into HEK293 cells, and after 48 h, relative luciferase activity was assessed.

Statistical analysis
Statistical analyses were conducted using Prism.All data underwent a normality test in Prism.For data with a normal distribution, Student's t-test and one-way analysis of variance were used, whilst the Mann-Whitney test was applied for non-normal data.Results were expressed as mean ± standard deviation (SD), with statistical significance determined at p < .05.

Hepatic stellate cell-specific deficiency of IGF2BP3 attenuates liver fibrosis in mice
IGF2BP3 cKO mice were utilised to investigate the role of IGF2BP3 in liver fibrosis (Figures 1C and 2A).Primary HSCs from these cKO mice were examined to assess the silencing efficiency of IGF2BP3 (Figure 1A,B).When fed a normal diet, the cKO mice exhibited no discernible abnormalities, and liver pathology and function were comparable to control mice (Figure S1A-S1E).
To determine the impact of IGF2BP3 deletion in HSCs on liver fibrosis progression, liver fibrosis models were established using CCl 4 and NASH.IGF2BP3 cKO mice treated with CCl 4 showed ameliorated liver fibrosis compared to their control littermates.IGF2BP3 knockout resulted in increased body and liver weight and reduced ALT/AST levels and collagen content (Figure 1D-K, Figure S1B).Additionally, IGF2BP3 cKO mice exhibited decreased levels of α-SMA and tissue inhibitor of metalloproteinases-1 (Timp1) (Figure 1G-K).Similarly, IGF2BP3 knockout inhibited liver fibrosis in the NASH mouse model (Figure 2).In conclusion, HSC-specific silencing of IGF2BP3 mitigated liver fibrosis in vivo.

Silencing IGF2BP3 induces hepatic stellate cell inactivation
To examine the impact of IGF2BP3 cKO on HSC activation status, primary HSCs were isolated from IGF2BP3  cKO mice.Compared to HSCs from control mice, those from IGF2BP3 cKO mice displayed a reduced rate of retinyl ester-positive intracytoplasmic lipid droplet loss (Figure 3A).Additionally, the levels of α-SMA, collagen type 1 alpha 1 (Col1A1), and Timp1, markers of HSC activation, were diminished (Figure 3B-E).Short hairpin RNAs were utilised to evaluate the effect of IGF2BP3 knockout on m 6 A levels and HSC inactivation.A significant reduction in the m 6 A content was observed in IGF2BP3deficient HSCs (Figure 4A and Figure S2A).HSC activation markers were downregulated in IGF2BP3-lacking HSCs (Figure 4B,C, Figure S2B and S2C).Furthermore, treatment with JQ1, a bromodomain and extraterminal domain inhibitor (BETi) of IGF2BP3, significantly attenuated HSC activation markers (Figure 4D-F and Figure S2D-S2F).These results indicate that inhibiting IGF2BP3 suppresses HSC activation.
In vivo, IGF2BP3 cKO mice exhibited higher levels of FPT indicators, such as iron, MDA, ROS, and depleted GSH, compared to NASH or CCl 4 model control mice.These levels were inhibited by Fer-1 (Figure S3A,D).Fer-1 also prevented the IGF2BP3 cKO-induced reduction in collagen levels (Figure S3B and S3E).Consistent with in vitro findings, GPX4 was downregulated in IGF2BP3 cKO mice (Figure S3,F).Collectively, these results suggest that inhibiting IGF2BP3 facilitates HSC FPT, at least in part, by suppressing GPX4.

IGF2BP3 deficiency-mediated m 6 A modification inactivates Jag1 signalling
To elucidate the mechanism by which IGF2BP3 deficiency mitigates liver fibrosis and suppresses HSC activation, m 6 A RIP-seq was conducted on isolated primary HSCs (Figure 6A).Downregulated genes in HSCs from IGF2BP3 cKO mice were predominantly enriched in the Notch pathway (Figure 6B).IGF2BP3 inhibition resulted in 16,949 hypomethylated sites and 961 hypermethylated sites (Figure 6C), highlighting IGF2BP3's critical role in HSC activation.Notably, Jag1, a hypomethylated transcript due to IGF2BP3 knockdown, emerged as the most downregulated gene in the Notch pathway (Figure 6C).Transcriptome analysis corroborated that Jag1 was significantly downregulated in the Notch pathway (Figure 6D).Thus, Notch-Jag1 signalling appears to mediate the effects of IGF2BP3-induced m 6 A modifications on HSC activation.Differentially expressed genes between the control and IGF2BP3 cKO groups were analysed to explore IGF2BP3's regulatory role, revealing downregulation of pro-fibrosisrelated genes (Figure 6E).Similar results were observed in LX2 cells following IGF2BP3 inhibition (Figure 6F).
Aberrant Notch signalling activation is often linked to its receptors and ligands.Silencing IGF2BP3 significantly reduced the expression of Jag1 and Notch3 (Figure 7A-C).Additionally, the protein levels of downstream molecules in the Notch-Jag1 signalling pathway, such as Hes1, Hes5, Hey1, and Hey2, were significantly decreased upon IGF2BP3 knockdown (Figure 7D,E).Immunofluorescence analysis confirmed that Jag1 and α-SMA levels were markedly attenuated after IGF2BP3 knockdown (Figure 7F).Similar reductions in Jag1, Notch3 and Hes1 levels were observed in LX2 cells post-IGF2BP3 inhibition (Figure S4).RIP-qPCR experiments demonstrated the direct binding between IGF2BP3 and Jag1 (Figure S4B).These findings implicate Notch-Jag1 signalling in the regulation of HSC activation by IGF2BP3 deficiency-mediated m 6 A modifications.

Jag1 overexpression suppresses IGF2BP3 KO-mediated ferroptosis of hepatic stellate cells
To further verify the role of Jag1 in counteracting the effects of IGF2BP3 cKO-induced m 6 A modifications on HSC activation, Jag1 was overexpressed, leading to elevated Jag1 lev-els in cells (Figure 8A-C).This overexpression increased Notch3 and Hes1 levels in primary HSCs (Figure 8D,E) and reversed the IGF2BP3 cKO-induced suppression of GPX4 (Figure 8E).Additionally, Jag1 overexpression mitigated the IGF2BP3 cKO-induced rise in FPT indicators, including iron, MDA, ROS, and depleted GSH (Figure 8F).Similar inhibitory effects on Notch signalling and HSC FPT were observed in LX2 cells following IGF2BP3 knockdown and subsequent Jag1 overexpression (Figure S5).These results indicate that the loss of IGF2BP3 inhibits HSC activation and promotes the FPT of HSCs by downregulating Jag1.

IGF2BP3 levels correlate with cirrhosis parameters in humans
Finally, the relevance of these findings to human pathology was investigated.qPCR analysis of liver specimens from patients with cirrhosis and healthy donors revealed significantly higher IGF2BP3 levels in cirrhotic patients (Figure S7A).Elevated IGF2BP3 levels were associated with increased liver injury and fibrosis, as indicated by Col1A1 expression (Figure S7B-D), suggesting that IGF2BP3 elevation may contribute to cirrhosis pathogenesis in humans.

DISCUSSION
The liver fibrosis development is primarily driven by HSC activation.Following injury, HSCs differentiate into activated myofibroblasts, which are responsible for the secretion and accumulation of fibrillar collagen.Therefore, inhibiting HSC activation represents a promising therapeutic strategy for treating liver fibrosis. 18The m 6 A modification of RNA plays a pivotal role in various physiological and pathophysiological processes in mammals. 19ur findings demonstrate that the loss of IGF2BP3 exerts an inhibitory effect on liver fibrosis, consistent with the results reported by Wang et al. 20 Whilst Wang et al. showed that IGF2BP3 loss leads to decreased HSC proliferation, the specific mechanisms were not fully explored.Our study suggests that knocking out IGF2BP3 promotes FPT of HSCs and inhibits their activation and proliferation, thereby preventing liver fibrosis progression.This high-lights IGF2BP3-induced m 6 A modifications as promising targets for liver fibrosis amelioration.Evidence increasingly supports the use of FPT to treat and prevent liver fibrosis. 21,22Wang et al. found that FPT in hepatocytes hinders liver injury recovery. 23Thus, inducing FPT in HSCs, rather than hepatocytes, presents a novel approach to treating liver fibrosis.IGF2BP3 inhibition was found to promote FPT in HSCs, primarily by damaging mitochondrial cristae.In vitro analysis of FPT-related factors such as GPX4, ACSL4, and SCL7A11 revealed that GPX4, an antioxidant enzyme that maintains ROS balance and negatively regulates FPT, plays a significant role in IGF2BP3 deficiency-induced FPT in HSCs.The effects of IGF2BP3 deficiency on FPT and GPX4 levels were notably suppressed by Fer-1, suggesting that IGF2BP3 deficiency promotes FPT in HSCs at least partially through GPX4.The m 6 A modification correlates with various human diseases and influences cell fate by affecting apoptosis, necroptosis, senescence and epithelial-mesenchymal transition. 24,25GF2BP3, a key m 6 A reader, significantly impacts gene regulation in human diseases.Yan et al. demonstrated that suppressing IGF2BP3 inhibits HCC cell migration and invasion, 26 whilst Deng et al. showed that IGF2BP3 loss induces FPT in glioblastoma by directly regulating GPX4 expression. 27Lu et al. established that IGF2BP3 can regulate HCC FPT. 7 However, its effect on HSC FPT remained unclear.This study established that IGF2BP3 downregulation and m 6 A are associated with HSC FPT.Although further research is necessary to fully elucidate m 6 A's influence on FPT, these results highlight a novel role for m 6 A in HSC FPT.Omics assays revealed that Jag1, a Notch signalling ligand, mediates the impact of IGF2BP3-induced m 6 A modifications on HSC activation.These results align with reports underscoring the critical role of Jag1 and Notch signalling in liver fibrosis. 28,29Upregulated Jag1 during HSC activation has been observed in both animal and human models, consistent with our findings. 29,30nterestingly, Jag1 promotes HSC activation by signalling through Notch1 and Notch3, rather than Notch2 or Notch4, reflecting the results of Kong et al. 28 Yang et al. demonstrated that HSC-specific Notch1 deletion in mice confers resistance to bile duct ligation-induced liver fibrosis. 31his study linked IGF2BP3 depletion to Notch-Jag1 signalling in HSC activation and liver fibrosis development.Antibodies targeting Jag1 and Notch1 have shown remarkable potential in treating N-Ras-induced primary liver cancer in mice. 32Utilising similar antibodies targeting IGF2BP3, Jag1, Notch1/3, and Hes1 as a therapeutic strategy in pre-existing liver fibrosis models would enhance the translational value of these discoveries.
The relationship between IGF2BP3-mediated Notch signalling and FPT in HSCs was further explored.qRT-PCR and Western blotting identified probable downstream signalling pathways associated with Jag1.IGF2BP3 knockdown significantly decreased the expression of Notch1/3 and Hes1.Notch1/3's essential role in liver pathophysiology is well-documented.Chen et al. reported activated Notch signalling in the CCl 4 model of liver fibrosis, marked by upregulated Notch3 and Hes1, consistent with our findings. 335][36] Chromatin immunoprecipitation and luciferase assays in this study suggested that Hes1 may directly bind to the GPX4 promoter, implying that Hes1 induces HSC FPT by binding to the GPX4 promoter, a key negative regulator of FPT.
Our study has several strengths and weaknesses.Using three methods to demonstrate that HSC IGF2BP3 knockdown attenuates liver fibrosis-short hairpin RNAs (shIGF2BP3), the BETi JQ1, and IGF2BP3 −/− miceenhances the reliability of our results.Despite significant discoveries, the study has limitations and unanswered questions.First, m 6 A, the most common internal RNA modification, is crucial in regulating the RNA metabolism of both protein-coding and non-coding RNAs. 37Hence, targeting m 6 A might inhibit HSC activation and liver fibrosis by regulating other protein-coding or non-coding targets.Second, other aspects of IGF2BP3's profibrotic effect in HSCs, such as inflammation signalling and microRNA release, require further investigation.Third, the CCl 4 mouse model showed increased liver and body weight following IGF2BP3 cKO, suggesting that IGF2BP3 deletion may also promote liver regeneration, a mechanism that needs further study.Lastly, incorporating human primary HSCs in experiments could provide better insights into the applicability of this mechanism in humans.
In summary, our data demonstrate that IGF2BP3 deficiency promotes HSC FPT and inactivation.Furthermore, targeting the IGF2BP3/Notch/Jag1 signalling axis presents a promising therapeutic approach for treating liver fibrosis.Developing specific antibodies against IGF2BP3 for future studies on liver fibrosis could significantly enhance the translational potential of our current findings.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no known financial conflicts of interest or personal relationships that could have influenced the findings reported in this paper.

F
I G U R E 1 HSC-specific knockout of IGF2BP3 alleviates CCl 4 -induced liver fibrosis.Primary HSCs liver, and serum were isolated from control and IGF2BP3 cKO mice.(A) qRT-PCR for IGF2BP3 in primary HSCs.(B) Western blot for IGF2BP3 in primary HSCs.(C) Illustration of the CCl 4 experimental design.(D,E) Liver weight and liver weight to body weight ratio, with representative gross appearance of livers (D).(F) Levels of ALT and AST.(G,H) HE staining, Sirius red staining, and immunohistochemical and immunofluorescence staining for α-SMA.Cell nuclei were stained with DAPI (blue) in immunofluorescence staining, as well as in subsequent similar experiments.(I-K) Expression of pro-fibrosis markers in primary HSCs.Data are presented as mean ± SD from six experiments.*p < .05,**p < .01,***p < .001.HSC, hepatic stellate cell; ALT, alanine aminotransferase; AST, aspartate aminotransferase; SD, standard deviation.

F
I G U R E 2 IGF2BP3 deficiency inhibits NASH-induced liver fibrosis.Primary HSCs, liver and serum were isolated from control and IGF2BP3 cKO mice.(A) Experimental design of NASH-induced liver fibrosis.(B) Body weight.(C) Representative gross appearance of livers.(D) Levels of ALT and AST.(E) Liver Triglyceride (TG) and Total Cholesterol (TC) content.(F) Liver weight and liver weight to body weight ratio.(G,H) HE staining, Sirius red staining, and immunohistochemical and immunofluorescence staining for α-SMA.(I-K) Expression of pro-fibrosis markers in primary HSCs.Data are presented as mean ± SD from six experiments.*p < .05,**p < .01,***p < .001.NASH, Non-alcoholic steatohepatitis; HSC, hepatic stellate cell; ALT, alanine aminotransferase; AST, aspartate aminotransferase; SD, standard deviation.F I G U R E 3 IGF2BP3 deficiency hinders the activation of primary HSCs and the expression of pro-fibrosis genes.(A) Primary HSCs were isolated from control and IGF2BP3 cKO mice and cultured in vitro for 1, 3 and 7 days.(A) Fluorescent photographs of BODIPY staining.(B,C) Immunofluorescence staining for α-SMA.(D,E) Expression of pro-fibrosis markers in primary HSCs.Data are presented as mean ± SD from three experiments.*p < .05,**p < .01,***p < .001.HSC, Hepatic stellate cell; BODIPY, boron-dipyrromethene; SD, standard deviation.

F I G U R E 4
Inhibition of IGF2BP3 blocks the activation and expression of pro-fibrosis markers in primary HSCs.Primary HSCs isolated from control mice were stably transfected with IGF2BP3 shRNA or treated with JQ1 for 24 h.(A) m 6 A levels.(B,C) Levels of IGF2BP3 and pro-fibrosis markers in HSCs with IGF2BP3 shRNA.(D,E) Levels of IGF2BP3 and pro-fibrosis markers in HSCs with JQ1 treatment.(F) Immunofluorescence staining of α-SMA and Type I collagen.Data are presented as mean ± SD from three experiments.HSC, Hepatic stellate cell; SD, standard deviation.

F I G U R E 6
IGF2BP3-mediated m 6 A modification regulates liver fibrosis by controlling Notch/Jag1 signalling.Primary HSCs were isolated from control and cKO mice and cultured in vitro for spontaneous activation.(A) m 6 A-RIP sequencing and RNA sequencing scheme.(B) Gene set enrichment analysis (GSEA) identifying Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with differentially methylated genes.(C) Methylation differences between control HSCs and IGF2BP3 cKO HSCs; peaks in green indicate hypomethylation, and peaks in red indicate hypermethylation.(D) Differential expression analysis between control HSCs and IGF2BP3 cKO HSCs; genes in green indicate low expression, and genes in red indicate high expression.(E) Heatmap of classical pro-fibrotic gene expression levels in primary HSCs.(F) Heatmap of classical pro-fibrotic gene expression levels in LX2 cells.HSC, Hepatic stellate cell.
Zheng: Designed the study.Xinmiao Li and Weizhi Zhang: Performed the majority of the experiments.Feng Jiang; Yining Wang and Lifan Lin: Analysed data and wrote the manuscript.Yifei Li; Lingling Wu and Han Zeng: Conducted revisions and added experiments.All authors contributed to the paper and approved the submitted version.A C K N O W L E D G M E N T SThe mechanism diagram was created using Figdraw (ID: ISOIOd79d7).The project was supported by the National Natural Science Foundation of China (No. 81873576) and the Wenzhou Municipal Science and Technology Bureau (No. Y20220023).